Pursuant to 37 C.F.R. 1.71 (e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
This invention pertains to the field of development of optimized genes that can render plants resistant to insects, nematodes, fungi, and other pests.
Genes coding for proteins with insecticidal activities are currently used in agriculture to control specific pests (Asgrow Reportsxe2x80x94Genetic Engineering for Pest Controlxe2x80x94Len Copping, Chapters 2.1-2.4). For example, genes coding for Bacillus thuringiensis (Bt) crystal proteins have been incorporated stably in several crops and are widely used as insect control agents (Pest. Sci. (1998) 52:165-175, Asgrow Reports, Supra.). Several other examples of different genes coding for insecticidal activity are also known (Asgrow Reports, supra.). However, the greatest limitation to using many of these genes is lack of sufficient activity (potency) and/or lack of useful spectrum of activity. For example, even the most widely used family of genes coding of crystal proteins are limited with respect to the pests they control and potency vs. various economically important pests (Asgrow Reports, supra.). For example, Bt toxins are weak versus corn root worms and other coleopteran pests.
Thus, a need exists for toxins that exhibit improved properties against various plant pests, and for methods of obtaining such toxins. Surprisingly, the present invention provides a strategy for solving each of the problems outlined above, as well as providing a variety of other features which will become apparent upon complete review of the following material.
The invention provides methods of obtaining an optimized recombinant pest resistance gene which can confer resistance to a pest upon a plant in which the gene is expressed. The methods involve (1) recombining a plurality of forms of a nucleic acid which comprise segments derived from a gene which can confer upon a plant resistance to a pest, wherein the plurality of forms of the nucleic acid differ from each other in two or more nucleotides, to produce a library of recombinant pest resistance genes; and (2) screening the library to identify at least one optimized recombinant pest resistance gene that exhibits improved pest resistance capability compared to a non-recombinant pest resistance gene.
In some embodiments, the methods also involve (3) recombining at least one optimized recombinant pest resistance gene with a further form of the pest resistance gene, which is the same or different from one or more of the plurality of nucleic acid forms of (1), to produce a further library of recombinant pest resistance genes; (4) screening the further library to identify at least one further optimized recombinant pest resistance gene that exhibits a further improvement in pest resistance capability compared to a non-recombinant pest resistance gene; and (5) repeating (3) and (4), as necessary, until the further optimized recombinant vector module that exhibits a further improvement in pest resistance capability compared to a non-recombinant pest resistance gene.
The invention also provides libraries that contain a plurality of recombinant pest resistance genes, wherein each recombinant pest resistance gene contains different permutations of segments of a gene which can confer upon a plant resistance to the pest.